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Recently, second harmonic generation (SHG) nanomaterials have been generated that are efficiently employed in the classical (NIR) and extended (NIR-II) near infrared windows using a multiphoton microscope. The aim was to test bismuth ferrite harmonic nanoparticles (BFO-HNPs) for their ability to monitor pulmonary macrophages in mice. BFO-loaded MH-S macro phages are given intratracheally to healthy mice or BFO-HNPs are intranasally instilled in mice with allergic airway inflammation and lung sections of up to 100 μM are prepared. Using a two-photon-laser scanning microscope, it is shown that bright BFO-HNPs signals are detected from superficially localized cells as well as from deep within the lung tissue. BFO-HNPs are identified with an excellent signal-to-noise ratio and virtually no background signal. The SHG from the nanocrystals can be distinguished from the endogenous collagen–derived SHG around the blood vessels and bronchial structures. BFO-HNPs are primarily taken up by M2 alveolar macro phages in vivo. This SHG imaging approach provides novel information about the inter action of macrophages with cells and the extracellular matrix in lung disease as it is capable of visualizing and tracking NP-loaded cells at high resolution in thick tissues with minimal background fluorescence.